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Chromosome looping at the human alpha-globin locus ismediated via the major upstream regulatory element (HS-40)

Citation for published version:Vernimmen, D, Marques-Kranc, F, Sharpe, JA, Sloane-Stanley, JA, Wood, WG, Wallace, HAC, Smith, A &Higgs, DR 2009, 'Chromosome looping at the human alpha-globin locus is mediated via the major upstreamregulatory element (HS-40)', Blood, vol. 114, no. 19, pp. 4253-4260. https://doi.org/10.1182/blood-2009-03-213439

Digital Object Identifier (DOI):10.1182/blood-2009-03-213439

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Published In:Blood

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RED CELLS, IRON, AND ERYTHROPOIESIS

Chromosome looping at the human �-globin locus is mediated via the majorupstream regulatory element (HS �40)Douglas Vernimmen,1 Fatima Marques-Kranc,1 Jacqueline A. Sharpe,1 Jacqueline A. Sloane-Stanley,1 William G. Wood,1

Helen A. C. Wallace,2 Andrew J. H. Smith,2 and Douglas R. Higgs1

1Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute for Molecular Medicine, University of Oxford, John Radcliffe Hospital,Oxford; and 2Institute for Stem Cell Research, University of Edinburgh, Edinburgh, United Kingdom

Previous studies in the mouse haveshown that high levels of �-globin geneexpression in late erythropoiesis dependon long-range, physical interactions be-tween remote upstream regulatory ele-ments and the globin promoters. Usingquantitative chromosome conformationcapture (q3C), we have now analyzed allinteractions between 4 such elementslying 10 to 50 kb upstream of the human �

cluster and their interactions with the�-globin promoter. All of these elements

interact with the �-globin gene in anerythroid-specific manner. These resultswere confirmed in a mouse model ofhuman � globin expression in which thehuman cluster replaces the mouse clus-ter in situ (humanized mouse). We havealso shown that expression and all of thelong-range interactions depend largelyon just one of these elements; removal ofthe previously characterized major regu-latory element (called HS �40) results inloss of all the interactions and �-globin

expression. Reinsertion of this elementat an ectopic location restores both ex-pression and the intralocus interactions.In contrast to other more complex sys-tems involving multiple upstream ele-ments and promoters, analysis of thehuman �-globin cluster during erythro-poiesis provides a simple and tractablemodel to understand the mechanismsunderlying long-range gene regulation.(Blood. 2009;114:4253-4260)

Introduction

Analysis of the human globin gene clusters and their associatedmutations that cause thalassemia have elucidated many of thegeneral principles underlying the regulation of mammalian geneexpression. In many gene clusters there is evidence that remoteregulatory elements are essential for high-level, tissue-specificexpression and that these elements physically interact with theircognate promoters. These findings pose the general questions ofhow such sequences interact with their promoters and how theyinfluence gene expression. In this study, we have evaluated thehuman �-globin cluster as a model to understand the mecha-nisms underlying long-range regulation of gene expression.

In all mammals studied to date, the �-globin genes appear tobe regulated by one or more of 4 remote, multispecies conservedsequences (MCS-R1 to R4) corresponding to erythroid-specificDNase1 hypersensitive sites (DHSs) lying upstream of thecluster.1 In the mouse �-globin cluster, we have previouslyshown that several upstream elements physically interact withthe �-globin promoters late in erythropoiesis as the �-globingenes are activated.2 However, orthologous upstream elementsmay play different roles in different species and the mouse doesnot appear to be an ideal model for the human � gene cluster.Deletion of the most highly conserved, orthologous element(MCS-R2) leads to almost complete down-regulation of human� gene expression but to only a modest reduction (�50%) inmouse �-globin synthesis.3 This suggests that other upstreamelements may interact and/or enhance �-globin expressionindependent of MCS-R2 in the mouse,3 possibly including a

hypersensitive site (HS �12) that has no human equivalent andmay be rodent specific.4

A better model is provided by a “humanized” mouse, inwhich 87 kb of the mouse � cluster is replaced in situ with117 kb of the human cluster. This replaces the mouse genes andregulatory elements with sequences that contain the humanelements and genes in their normal relative positions. Thehuman MCS-R1 to -R4 correspond to 4 DHSs (HS �48,HS �40, HS �33, HS �10; Figure 1A), of which MCS-R2(HS �40) appears to be the major remote, distal enhancer ofadult � gene expression.5 The influence (if any) of the otherelements (HS �48, HS �33, and HS �10) is not clear.1 Here wehave assessed in detail the physical interactions of theseelements with each other and with the �-globin promoter andshown similar interactions in both normal primary humanerythroblasts and erythroblasts from the humanized mouse.Furthermore, we demonstrate that in the absence of HS �40 inthe mouse model, in which there is no � gene expression, noneof the remaining upstream elements interacts with the �-globinpromoter. When HS �40 is reinserted at an ectopic site 3� to the� genes, both the interactions and expression are reinstated.Therefore, in the human �-globin cluster both physical interac-tion and enhancement of expression appear to depend predomi-nantly on a single upstream element (MCS-R2/HS �40). Thisprovides a relatively simple model for addressing the generalquestion of how remote upstream regulatory elements interactwith their cognate promoters in vivo.

Submitted March 30, 2009; accepted August 1, 2009. Prepublished online asBlood First Edition paper, August 20, 2009; DOI 10.1182/blood-2009-03-213439.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2009 by The American Society of Hematology

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Figure 1. Intrachromosomal interactions, between the human �-globin genes and conserved upstream elements. (A) Chromosomal organization of the human �-globin locusannotated as previously described.23 Red numbers indicate the points (coordinates) analyzed by q3C. q3C assays were performed using HindIII-digested, fixed chromatin from primaryT lymphocytes (gray bars) and primary erythroblasts (red bars), generating restriction fragments of 3 to 5 kb. This allowed us to use q3C to evaluate all interactions between the �-globingenes and a variety of points along the �-globin cluster, including all the remote upstream elements. Twelve restriction fragments located within (HS �48, HS �46, HS �40, HS �33, andHS �10 and the �-globin genes) or outside (fragments 53, 189, 218, 380, 401, and 441) the �-globin locus were chosen to determine the locus conformation (A). The shaded areacorresponds to the region containing all sequences that interact with the �-globin genes (B-J). The bar chart (y-axis) shows the enrichment of PCR product (%) normalized to theenrichment within the human Ercc3 gene (� 100%). This provides an internal, genomic control for the cross-linking procedure and any general changes in nuclear or chromatin structure.24

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Figure 1 (continued). These independent graphs represent a measure of the association between the points indicated (x-axis) to the anchor points (B) “fragment 53,” (C) HS �48,(D) HS �46, (E) HS �40, (F) HS �33, (G) HS �10, (H) �-globin genes, (I) “fragment 189,” and (J) “fragment 380.” For each new anchor point, a new Taqman probe and reverse primerwere designed and thus results can be compared only within, but not between, each set of graphs. The resolution of this 3C assay can be estimated by comparing “positive” and “negative”restriction fragments located at a similar distance to their target. For example, the locus position 189 is located 26 kb downstream of �-globin and does not interact, whereas HS �10,located 26 kb upstream of �-globin, shows a 3-fold enrichment in erythroid cells (H). Data shown represent the average of a least 3 independent experiments using Taqman/real-time PCR.Error bars denote SEM. Each PCR was performed several times and averaged. Signals were normalized to the total amount of DNA used, estimated with an amplicon located within aHindIII fragment (Table 1). Coordinates of the points analyzed are indicated on the x-axis.

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Methods

Primary cells and cell lines

Human erythroid progenitors were derived from peripheral blood mono-nuclear cells of healthy subjects and grown in 2-step cultures.6,7 Humanactivated T lymphocytes were obtained from buffy coats by culture for3 days in RPMI/20% fetal calf serum in the presence of 2% phytohemagglu-tinin M and 20 U/mL interleukin-2. Mouse primary erythroid cells wereobtained from the spleens of acetylphenylhydrazine-treated mice,8 purifiedfor Ter119-positive cells by auto–magnetic-activated cell sorting; Ter119-negative cells from the same source served as a nonerythroid control.

Chromosome conformation capture

The chromosome conformation capture assay (3C) was performed asdescribed previously using the same set of controls.2 Human and mouseprimers and probes used are shown in Table 1.

ES cell genetic manipulation and generation of humanizedmouse models

The generation and characterization of the humanized mouse model withfunctional human �-globin genes and a second model with HS �40 deleted havebeen previously described.9 To create a mouse model in which the HS �40 waspositioned at an ectopic location, the previously described BAC clone containingthe human �-globin regulatory domain with the HS �40 deletion (supplementalFigure 1A, available on the Blood website; see the Supplemental Materials link atthe top of the online article) was further engineered by recombineering inEscherichia coli DY380 cells. This enabled the insertion of a cassette consistingof a PGK-zeocyin resistance gene (flanked by frt-F5 recombination sites) linkedto a 1.1-kb HS �40 sequence into a position downstream of the �-globin genes(sequence position 169470 mapped on HG18). This newly modified BAC clonewas then co-electroporated with a Cre expression plasmid into the double-targeted mouse embryonic stem (ES) cell line 2/H-1 with loxP and lox511 sites atthe Dist (c16orf8) and theta (HbQ) genes. Cre recombination, as previouslydescribed,9 results in genomic replacement of the mouse sequence by humanBAC sequence between positions delineated by loxP and lox511 sites andreconstruction of a hypoxanthine phosphoribosyl transferase (Hprt) minigene toallow selection of cells in medium containing hypoxanthine, aminopterin, andthymidine (HAT) (supplemental Figure 1B). From one electroporation, a singleHAT-resistant clone was obtained that was also resistant to zeocyin andpuromycin as expected. Cells of this HAT-resistant clone were subsequentlyelectroporated with a FLPe expression vector to excise the Hprt, puromycin, andzeocyin resistance genes via their flanking frt, frt-F3, and frt-F5 sites, respectively(supplemental Figure 1B). Hprt-negative cells were selected in 6-thioguanine andresistant clones recovered at high frequency that were also puromycin sensitiveand zeocyin sensitive. One such clone was checked to confirm the expected DNAsequence rearrangements. This was subsequently used to generate chimeras.Male chimeras were backcrossed to C57BL/6 females and agouti offspringgenotyped to confirm germline transmission.

Genomic DNA analysis across the entire humanized insert and RNaseprotection assays were performed as previously described.9 In addition, the3�HS �40 construct was also analyzed by sequencing and by polymerase chainreaction (PCR) using an amplicon spanning the junction of insertion (primersequences: Fw, GCAATATCCGCTACAGCCAAAT; Rev, GAGCTCTT-GGGCAGAGATATGG; probe, CGGATCCCCCAACAAGAAAACCAGC).Study protocols received ethical approval from Oxford University EthicalReview Panel and all animal work was carried out under Home Office License.

Results

3C analysis of regulatory elements and promoters in thehuman �-globin cluster

Here we have analyzed, for the first time, all interactions between thehuman �-globin regulatory elements and the �-globin promoters in

primary human erythroid and nonerythroid cells using a quantitative 3Cassay (q3C).2 HindIII restriction fragments containing HS �48, HS �46,HS �40, HS �33, HS �10, and the � genes, or regions outside the�-globin locus, were chosen to determine the locus conformation(Figure 1A). We initially evaluated q3C between the � genes and

Table 1. Oligonucleotide primers and probes (q3C)

Designation Sequence

Human primers

53 H3 P TTTCTGGAGGAGGACACAACTGATTTCCC

HS48 H3 P CAGGCCAAACATAGCAACACCATCCC

HS46 H3 P AGTCTAGTCCCCTCAGCTTTTAGCAT

HS40 H3 P CCTGATTGCTAAAGCCTTTTCACAGCTGG

HS33 H3 P CAAGATGTGCAAGCCCACTGTGAT

HS10 H3 P TGGTCTTTTGCGTTTGGTGACAGTCAA

a2 H3 P CGGGAGCGATCTGGGTCGAGG

189 H3 P TCTCTCGGATCTGAATGAACCCCAAGTG

380 H3 P TCAATGCAAAGAAAACCATAGCCAATT

153 H3 R CACTATATTGGAAGACCATGCAACTC

53 H3 A F AAAGAAGGATGTGTCCAGCTCATC

HS48 H3 A R AAGTGGACTGTGAAAGAAATAAGGAAA

HS46 H3 A R TCACAGGTCCTGTCTAGTTCTAAGGTT

HS40 H3 A R CATAAAGACACCACATGCACTAGCT

HS33 H3 A R CTGGTATTTCTGCCATTTGGAAA

HS10 H3 A F AACAGATGCCAGCCAAACAGA

189 H3 A R ACCTTGGTCTCCATGACAATGAC

380 H3 A R AGTTCGTTTTCCAGCAGTTTGACTA

a2 H3 A R GAGAGGAAGGCGCCATCTC

53 H3 F AATCAGTTGTGTCCTCCTCCAGAA

HS48 H3 R AATAAGGAAACTGGGATGGTGTTG

HS46 H3 R GATGCTTTCTCAGATGCTAAAAGCT

HS40 H3 F AGCTGTGAAAAGGCTTTAGCAATC

109 H3 F CCCTTATCCCAAACACTTAAACTTTC

HS33 H3 R CTGGTATTTCTGCCATTTGGAAA

HS10 H3 F GACTGTCACCAAACGCAAAAGAC

a2 H3 R GCCGCTCACCTTGAAGTTGA

189 H3 R ACTTCGGTGGCAAGTTACACTTG

218 H3 R ACAGTCTTCGATAGAGTTTGAAAGCTT

380 H3 R TCTTTGCATTGATTCTTGTTAAGCTT

401 H3 F GCGATGACGCGGCACTT

441 H3 F AAACAGCCAAGTGGATACATTCAA

xpb H3 F CGGTGAGGTGAGTTTGTGGAAT

xpb H3 R AGGATCTCTGTTTAATGGAAAAGCTT

xpb H3 P AAGGATGAAGGCGTGATCCGACTCTG

hPr/Ex1 F GGGCCGGCACTCTTCTG

hPr/Ex1 R GGCCTTGACGTTGGTCTTGT

hPr/Ex1 P CCCACAGACTCAGAGAGAACCCACCATG

Mouse primers

24 H3 F ATGGGACAATGGAGGGCTAAG

55 H3 R TCTATTAGGGATGGCACTTTACGAA

72 H3 R CCCCTGTGGGAAAGACAGAAA

78 H3 F AGACAAGGAAATGGAAGAGACATCA

HS31 H3 F AGCTGTGCCTGGCAGATGAC

HS29 H3 F AGGAAGAAGAGGTTCAACAAGCAT

HS26 H3 R GAATCTCCATCTCCAAGGG

Zeta H3 F ACAACGTGGAGAGGACTGAGAGAT

174 H3 F AATGGTTGCCTTTTAAAGACATTTGT

a2 H3 R GTGGCCATGAGCCAAAGAAT

a2 H3 P CCTCCAGGTACCATAAATCTCAGACTTG

xpb H3 F CTTCTGCTACAAAGCGCTGACTT

xpb H3 R TGCCCTCCCTGAAAATAAGGA

xpb H3 P TGCACCCTGCTTTAGTGGCCTTGTAGG

mPr/Ex1Fw TGGAGGGCATATAAGTGCTACTTG

mPr/EX1Rev TGCTTTTGTCTTCCCCAGAGA

mPr/Ex1P TGCAGGTCCAAGACACTTCTGATTCTGACA

Oligonucleotide sequences used in real-time PCR.P indicates probe; R, reverse; A, anchor; and F, forward.

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various fragments along the �-globin cluster (Figure 1H), demonstratingthat the human � genes specifically interact with the upstream elementsin erythroid cells. They also interact with HS �46, which is not anerythroid element but corresponds to a constitutive HS that binds CTCFin all tested cell types (Marco de Gobbi, unpublished data, October2009). These data demonstrate that despite differences in the functioningand positions of these elements in human and mouse clusters, the overallarchitecture of the interactions is maintained in the 2 species.2

To examine these interactions in detail, each restriction frag-ment was used as a bait (anchor fragment) in q3C experiments,using a unique Taqman probe for each fragment. In total, 120 primercombinations have been used across 400 kb of DNA encompassingthe �-globin gene cluster (Figure 1A). Regions located outside theglobin locus do not interact with the � cluster either in erythroblastsor T lymphocytes (Figure 1B,I-J). By contrast, all fragmentscontaining the remote regulatory elements and the �-globin genescome into close spatial proximity with each other in erythroblasts,whereas such interactions are relatively weak (or absent) inT lymphocytes (Figure 1C-H). Not all elements interact equally.For example, little interaction was seen between HS �40 andHS �48 in erythroid cells (Figure 1C,E).

3C analysis of regulatory elements and promoters in thehumanized mouse

Having determined the interactions within the cluster in normalhuman erythroblasts, we determined whether these were replicated

in the humanized mouse (Figure 2A). Expression of the human �genes is approximately 50% that of the mouse genes in these mice.9

Using q3C, these interactions were analyzed in erythroid cells fromnormal humanized mice, both in homozygotes (supplementalFigure 2) and heterozygotes (Figure 2C). This showed exactly thesame pattern as seen in primary human erythroblasts (compareFigure 2C with Figure 1H). The control mouse cluster shows theexpected interactions between the upstream elements with thepromoters (Figure 2D) as seen previously.2 (Note that HS �10could not be analyzed because of a SNP located at a HindIII site inthese mice.) This humanized mouse therefore provides a suitablemodel to study this aspect of human �-globin regulation.

Interactions when HS �40 is deleted

We next studied humanized mice (heterozygotes) in whichHS �40 had been deleted (�HS �40). In these mice, �-globinexpression is severely down-regulated to less than 2% of mouse� gene expression9 (see also Figure 5). Given such changes ingene expression (which may also be associated with changes inchromatin accessibility), it was important to assess the cleavageof chromatin by endonuclease (HindIII) at critical sites becausechanges in the 3C assay might reflect changes in endonucleaseaccessibility rather than looping. Using a quantitative PCRassay, we demonstrated equal HindIII cleavage efficiency atcritical sites in normal cells and cells from which HS �40 hadbeen deleted (supplemental Figure 3).

Figure 2. Deletion or ectopic reinsertion of HS �40 influences the interactions between the remote HS and the �-globin genes. Intrachromosomal interactionsinvolving human remote regulatory sequences and the human �-globin genes in normal, �HS �40, and 3�HS �40 humanized alleles. (A) Chromosomal organization of thehumanized �-globin locus. (B) Chromosomal organization of the mouse �-globin locus. Red and blue numbers indicate, respectively, the points (coordinates) of the human andmouse primers analyzed by q3C. q3C assays were performed using HindIII-digested, fixed chromatin from primary Ter119� (blue graph bars) and Ter119� (red graph bars)cells from spleens obtained from phenylhydrazine-treated mice. The bar chart (y-axis in panels C-H) shows the enrichment of PCR product (%) normalized to the enrichmentwithin the mouse Ercc3 gene (� 100%), and thus explains the scale difference compared with Figure 1, normalized on human Ercc3. These independent graphs represent ameasure of the association between the remote regulatory sequences (x-axis) to the �-globin genes (anchor fragment) in normal-humanized locus (C), endogenous mouselocus (D), �HS �40 humanized locus (E), endogenous mouse locus (F), 3�HS �40 humanized locus (G), and endogenous mouse locus (H). Results obtained withendogenous mouse locus are in agreement with those observed previously.2 Data are represented as in Figure 1. To the left are images representing the proposedconfigurations interpreted from these data.

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The chromosome conformation assumed in the absence ofHS �40 is shown in Figure 2E. In the absence of HS �40, nostable interactions occur between the remaining upstream elements(HS �48, HS �46, and HS �33) and the � genes. This indicatesthat HS �40 plays a crucial role in mediating the long-rangeinteractions between the upstream elements and the � promoter. Toensure that the changes in chromosome looping were not simplydue to differences in the stages of erythroid maturation betweenexperiments, this was assessed by morphology and fluorescence-activated cell sorting analysis (Figure 3). As a further control, wehave shown that interactions at the endogenous mouse �-globincluster (the other allele in humanized mouse heterozygotes) areintact in all situations (Figure 2F).

The effect of reinserting HS �40 at an ectopic site

Removal of HS �40 abolished all interactions. To determine whetherthese interactions could be restored by HS �40 alone, a humanized

mouse was generated from ES cells with HS �40 reinserted into the �cluster, but at an ectopic site, 3� to the � genes (3�HS �40, Figure 4A).To accomplish this, a previously described9 BAC clone, containing thehuman �-globin regulatory domain from which HS �40 had beendeleted, was further modified by recombineering to insert a 1.1-kbHS �40 sequence linked to this new position in the same chromosomalorientation as found at its natural site (Figure 4A). This newly modifiedBAC clone was then used for Cre recombinase–mediated genomicreplacement of the mouse �-globin synteny region using the ES cell lineas previously described.9 An ES clone was obtained after selection withthe correct replacement and this was subsequently modified by a finalround of recombination using FLP recombinase to remove all selectionmarker cassettes (Figure 4 and supplemental Figure 1B). An ES cellclone now containing HS �40 at the ectopic position with minimalextraneous DNA sequences was checked extensively to confirm thecorrect sequence rearrangements were present, and used to establish thenew humanized line (3�HS �40).

Figure 3. Erythroid cells analyzed in this study are all atsimilar stages of differentiation. (A) May-Grunwald Giemsa(MGG)–stained touch preparations from the spleens ofphenylhydrazine-treated adult mice. Images were captured usinga Nikon eclipse E600 microscope with a Nikon DXM1200C digitalcamera using NIS elements BR2.30 SP4 imaging software (allfrom Nikon UK). Original magnification 60�/1.40 NA oil objective(Nikon Japan) for all panels. (B) Flow cytometric analyses of mouseprimary erythroblasts. Ter119� erythroid cells were auto–magnetic-activated cell sorting–purified from the spleen of phenylhydrazine-treated adult mice and restained with anti-CD71 to show theequivalent maturation stages in all mice examined.

Figure 4. Humanized chromosomes used to study effect of ectopic reinsertion of HS �40. (A) An outline of the 3 recombinant humanized chromosomes analyzed in thisstudy: (top) humanized chromosome with intact normal sequence in which HS �40 is located within the C16orf35 gene (referred to as normal humanized chromosome);(middle) humanized chromosome with HS �40 deleted from the C16orf35 gene (referred to as �HS �40 chromosome); (bottom) humanized chromosome with HS �40 deletedfrom the C16orf35 gene and relocated to an ectopic position (referred to as 3�HS �40 chromosome). Human genes are represented by gray filled rectangles; mouse genes, byopen rectangles. Human and mouse sequences have boundaries in Dist and theta genes indicated by m/h and h/m. Human/mouse boundaries and positions of deletion andreinsertion of HS �40 correspond to the position of site-specific recombination sites (key) used to engineer these rearrangements. For a more detailed explanation of thechromosome engineering steps involved, see supplemental Figure 1A and B. (B) Southern blot analyses of the HS �40 reinsertion site with a PstI � probe that hybridizes withthe �-globin genes. Genomic DNA was digested with BglII and HindIII and run on a 0.65% agarose gel. From left to right: genomic DNA from mouse, human, humanized micelacking HS �40, normal humanized mouse, and with the reinsertion of HS �40 at the 3� end of the cluster. Arrowheads show a size increase (1.1 kb) in the genomic DNAfragment of mice with the reinsertion of HS �40 between the �-globin gene and the gene. The normal 9.4 kb (open arrowhead) BglII fragment is increased to 10.5 kb (closedarrowhead) and the normal 4.1 kb (open arrowhead) HindIII fragment is increased to 5.2 kb (closed arrowhead). A small restriction map is represented at the bottom of panel A.

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The humanized locus was analyzed by Southern blot (Figure4B), as previously described,9 with probes spanning the entirehumanized locus and its insertion sites. Junctions at the HS �40reinsertion sites were checked by PCR amplification and subse-quent sequence analysis.

After reinsertion, �-globin RNA transcription occurs to approxi-mately 50% of that seen when HS �40 is in its normal position (Figure5).As HS �40 now lies in the same HindIII restriction fragment as the �genes themselves (Figure 4B), the HS �40/promoter interaction couldnot be examined. But, significantly, all the remaining upstream se-quences again interacted normally with the anchor fragment (Figure2G). Thus, in the presence of HS �40, albeit in a different position, theother upstream elements can once more interact with the � genepromoters and expression occurs, although this appears to be atsuboptimal levels. Again, the endogenous mouse cluster is unaffected(Figure 2H).

Discussion

This study represents an important step toward understanding how thepreviously characterized remote upstream control elements (MCS-R1 to-R4) interact with the �-globin promoters to control gene expression.This in turn provides a good model for understanding, in general, howdistal elements control mammalian gene expression. Our observationsshow that in humanized erythroid cells the physical interactions betweenall of the upstream elements and the �-globin promoters depend largelyon a single element (MCS-R2), a 1.1-kb HpaI fragment containingHS �40 (Figure 4A). Deletion of this remote upstream element resultsin almost complete lack of expression ( 2% of normal) and of all theintrachromosomal interactions. However, when MCS-R2 is re-inserted, now downstream of the � cluster, it restores both of thesefeatures (Figures 2-3).

At present, the mechanism by which remote regulatory ele-ments and their cognate promoters interact is still not clear,although several models (looping, tracking, facilitated tracking,and linking) have been proposed.10 Many observations havesupported the idea that chromatin loops result from the stabilizationof random interactions between cis-elements in chromatin. Theobservations made here are also most consistent with a loopingmodel in which multiprotein complexes at the upstream element(MCS-R2) and the � gene promoters collide, and their interactionsbecome stabilized, in erythroid cells (Figure 2). This in turn mightnucleate interactions with other upstream elements (MCS-R1/HS �48 and MCS-R3/HS �33). Certainly, the establishment ofthese interactions is not entirely dependent on the position of

HS �40, which activates �-globin from its natural location (up-stream) and from its relocated position (downstream) of the cluster.

The true role of the other MCS-R elements is not clear. To date,there are no natural deletions that remove these elements withoutalso removing HS �40.5 Furthermore, we and others have ob-served no major effect of these elements on expression intransgenic experiments.11-13 Nevertheless, because interactionswith HS �48 and HS �33 are present when MCS-R2 is reinserted,these sites may yet be relevant to the control of �-globin geneexpression. For example, they may in some way facilitate theinteraction between HS �40 and the �-globin genes. It is thereforeinteresting to note that when MCS-R2 is reinserted downstream ofthe cluster, human �-globin expression is suboptimal (� 50% ofthat seen when MCS-R2 is located upstream), suggesting that itsnormal chromosomal position may be important for fully efficientactivation. Together, these observations emphasize the need toanalyze, in greater detail, the role of each individual regulatoryelement, and the importance of the position and orientation ofMCS-R2 with respect to the �-globin cluster.

The MCS-R2 fragment contains binding sites for the GATA-1/SCL complex, NF-E2, and Sp/XKLF.2,14 It has been establishedthat in the �-globin locus various transcription factors (GATA-1,EKLF), cofactors (Idb-1, a component of the GATA/SCL com-plex), and chromatin remodeling complex (BRG1, a component ofthe Swi/Snf complex) may mediate physical interactions betweenerythroid promoters and upstream elements.15-18 However, simplebinding and interaction of GATA-1 or ldb-1 could not fully explainthis phenomenon at the �-globin locus because these factors arerecruited during the early phases of erythropoiesis, before loopingoccurs.14,19 However, Sp/XKLF factors are recruited, at the up-stream elements and promoter late in erythropoiesis, when loopingoccurs.2 But, again, it is unlikely that a single member of this familycould mediate looping because many of the upstream elements arebound by Sp/XKLF factors and yet these upstream elements do notinteract with the �-promoters in the absence of HS �40. Furthermore, apreviously described knockout of EKLF apparently had little effect on�-globin expression.20 It has also been suggested that PolII may play arole as a bridging factor between upstream elements and their cognatepromoters.21 However, HS �48 still binds PolII in the absence ofHS �40 and yet this element does not appear to interact with or facilitatesignificant recruitment of PolII to the �-globin promoters on its own.Furthermore, looping at the �-globin cluster persists in the absence ofPolII, after �-amanitin treatment.22 Thus, the proteins and mechanismsmediating interactions between the upstream elements and their promot-ers are still poorly understood.

This study shows that both enhancer activity and physicallooping in the human �-globin cluster predominantly depend on asingle cis-acting element (MCS-R2/HS �40) in contrast to themore complex cis-acting interactions observed in gene clusters(including the human and mouse �-globin clusters and the mouse�-globin cluster) involving multiple cis-acting elements. This fact,together with the development of the humanized mouse system,now provides a tractable, in vivo experimental model to understandin detail some of the issues raised in this and other studiesconcerning the mechanism by which mammalian gene expressionis controlled via distal regulatory elements.

Acknowledgments

We are very grateful to Richard Gibbons and David Garrick forcritically reading the paper. This work was funded by the Medical

Figure 5. �-globin expression in humanized mice. RNase protection assay ofadult blood RNA from heterozygotes of lines �HS �40, 3�HS �40, and normalhumanized mouse line.

CHROMOSOME LOOPING AT THE HUMAN �-GLOBIN LOCUS 4259BLOOD, 5 NOVEMBER 2009 � VOLUME 114, NUMBER 19

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Research Council and the National Institute for Health Research(NIHR) Biomedical Research Center Program.

AuthorshipContribution: D.V. designed and performed research, analyzeddata, and wrote the first draft of the paper; F.M.-K. designed andperformed research and analyzed data; J.A.S., J.A.S.-S., andW.G.W. provided samples and maintained the animal stocks;

H.A.C.W. and A.J.H.S. designed and generated the humanizedmodel with ectopic HS �40 reinsertion; and D.R.H. designedresearch, analyzed data, and wrote the final draft of the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Doug R. Higgs, MRC Molecular HematologyUnit, Weatherall Institute of Molecular Medicine, John RadcliffeHospital, Headington, Oxford, OX3 9DS, United Kingdom; e-mail:doug.higgs@imm.ox.ac.uk.

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